Enzyme linked Immuno-Sorbent Assay (ELISA)

(Principles and Protocols)

What is ELISA ?Enzyme Linked Immuno-Sorbent Assay (ELISA) is a biochemical assay technique or Serology based detection used mainly in Immunology.It is a plate-based assay designed for detecting and quantifying substances such as peptides, proteins, antibodies & hormones.Advantages :1.Sensitivity of virus diagnosis.2.Cost effective.3.A large number of samples can be tested for virus infection.Disadvantages :Detection of virus of unknown origin is not possible by serology as well as its inability to detect antigenic property is not functional for viroid (absence of coat protein).

ELISA : Sensitive and efficient Good for large scale testing Ready to use test-kits available commercially Dependent on good antisera (expensive)

Negative result Positive result Microtiter well

Fig : ELISAplate

Fig : ELISA Detector

Principles of ELISA :

The sensitivity of detection depends on amplification signal during the analytic reaction. In some enzymatic reaction, the signal generated by enzyme which are linked to the detection reagents in fixed proportions to allow accurate quantification (enzyme linked).

There are two main variations on ELISA method is :

1. ELISA can be used to detect the presence of antigens that are recognized by an antibody or

2. It can be used to test for antibodies that recognize an antigen.

Types of ELISA :

Qualitative ELISA : Positive (+ ve) or Negative (- ve ) results.

Quantitative ELISA : Optical density or fluorescent units of the sample is interpolated into a standard curve, which is typically a serial dilution of the target.

ELISA Methods :

Direct ELISA

Indirect ELISA

Double Antibody Sandwich ELISA ( DAS )

Ag = Antigen

Direct ELISA DAS ELISA Indirect ELISA

Antibody Structure :Antibodies are immune system-related proteins called immunoglobulins. Each antibody consist of 4 polypeptide chains and 2 light chains joined to form a ‘ Y ’ shaped molecules.

The amino acid sequence in the tips of the ‘ Y ‘ varies greatly among different antibodies. This variable region, composed of 110-130 amino acids, give the antibody its specificity for antigen binding.

The variable region includes the ends of the light and heavy chains.

The constant region determines the mechanism used to destroy antigen. Antibodies are divided into 5 major classes : IgM, IgE, IgG, IgA and IgD based on their constant region structure and immune function.

Antigen(Ag)-Antibody(Ab)

Reaction :

Antibodies :

Polyclonal Antibody Monoclonal Antibody

1.Derived from different B- lymphocyte cell lines.

1.Derived from a single B- cell clone.

2.Batch to Batch variation affecting Ab reactivity & treatment.

2.No Batch to Batch variations. Effectiveness of Ab is much more predictable.

3.NOT Powerful tools for clinical diagnostic tests.

3.Enable the development of secure immunoassay systems.

Direct ELISA : Protocols : Direct ELISA is suitable for the detection of proteinaceous antigens and may require pre-purifications of sample. Direct ELISA can be performed when desired antibody is available in a pre-conjugated state (fluorometric, colorimetric, enzymatic). Advantages :1. Fast2. Eliminates possible non-specific binding of secondary antibody Disadvanatges :1. Reactivity of primary antibody may be reduced conjugation2. Little signal amplification

Indirect ELISA : Protocol : If the primary antibody is not conjugated, then indirect ELISA is required in which a conjugated secondary antibody is targeted to the isotype ( e.g. mouse IgG1, goat IgM, chicken IgY etc.) of the primary antibody. Advantages :1. Wide variety of secondary conjugates are available for detection.2. Immuno-reactivity of the primary antibody is not compromised.3. Multiple binding of the secondary affords some signal amplification. Disadvantages :1. Extra step in the protocol.2. Some non-specific binding of the secondary may cause high risk.

Double Antibody Sandwich (DAS) ELISA :

Add Antibody in the microtiter well

A. Add Sample / virus + ve

B. Incubation and Washing

C. Addition of Enzyme labelled antibody

D. Incubation and Washing

E. Addition of Substrate ( Hydrogen peroxide+Diamino benzene)

G. Incubation

H. Brown color in virus + samples

Fig : Comparison of Different types of ELISA